Differential solubilization of membrane lipids by detergents: coenrichment of the sheep brain serotonin 5-HT1A receptor with phospholipids containing predominantly saturated fatty acids

It is a widely held belief that the inactive X-chromosome (Xi) in female cell nuclei is strongly condensed as compared to the largely decondensed active X-chromosome (Xa). We have reconsidered this problem and painted X-chromosome domains in nuclei of subconfluent, female and male human amniotic fluid cell cultures (46,XX and 46,XY) by chromosomal in situ suppression (CISS) hybridization with biotinylated human X-chromosome specific library DNA. FITC-conjugated avidin was used for probe detection and nuclei were counterstained with propidium iodide (PI). The shape of these nuclei resembling flat ellipsoids or elliptical cylinders makes them suitable for both two-dimensional (2D) and three-dimensional (3D) analyses. 2D analyses of Xi- and Xa-domains were performed in 34 female cell nuclei by outlining of the painted domains using a camera lucida. Identification of the sex chromatin body in DAPI-stained nuclei prior to CISS-hybridization was confirmed by its colocalization with one of the two painted X-domains. In 31 of the 34 nuclei the area AXi for the inactive X-domain was smaller than the area AXa for the active domain (mean ratio AXa/AXi = 1.9 +/- 0.8 SD, range 1.0-4.3). The signed rank test showed a highly significant (P < .0001) difference both between AXa and AXi and between the ratios r(Xa) and r(Xi), calculated by dividing the maximum length L of each X-domain by its maximum width W. In most nuclei (26/34) we found r(Xa) > r(Xi) demonstrating a generally more elongated structure of Xa. For 3D analysis a confocal scanning laser fluorescence microscope (CSLFM) was used. Ten to 20 light optical sections (PI-image, FITC-image) were registered with equal spacings (approx. 0.4 microns). A thresholding procedure was applied to determine the PI-labeled nuclear and FITC-labeled X-domain areas in each section. Estimated slice volumes were used to compute total nuclear and X-domain volumes. In a series of 35 female nuclei most domains extended from the top to the bottom nuclear sections. The larger of the two X-chromosome domains comprised (3.7 +/- 1.7 S.D.)% of the nuclear volume. A mean ratio of 1.2 +/- 0.2 SD (range 1.1-2.3) was found for the volumes of the larger and the smaller X-domains in these female nuclei. In a series of 27 male amniotic fluid cell nuclei the relative X-chromosome domain volume comprised (4.0 +/- 2.6 S.D.)%.(ABSTRACT TRUNCATED AT 400 WORDS)     

Theoretical Approach to Optimization of Home Subscriber Server in IP Multimedia Subsystem

Wireless Personal Communications - Our paper deals with structure of Home Subscriber Server in IP Multimedia Subsystem and mathematical models for optimizing this database. The aim of our work is...     

"Repty" scales of strokes. Functional index "Repty" for the evaluation of ADL in hemiplegic patients after cerebral stroke. Part II

The Escherichia coli FtsH protein is a membrane-bound and ATP-dependent protease. In this study, we describe ATP-dependent conformational changes in FtsH as well as a polypeptide binding ability of this protein. A 33 kDa segment of FtsH became trypsin resistant in the presence of ATP. ATP and ATPgammaS prevented self-aggregation of detergent-solubilized FtsH-His6-Myc at 37 degrees C, again suggesting that the binding of ATP induces a conformational change in FtsH. Affinity chromatography showed that FtsH-His6-Myc can associate with denatured alkaline phosphatase (PhoA) but not with the native enzyme. Denatured PhoA also prevented the aggregation of FtsH, and these two proteins co-sedimented through a sucrose gradient. Binding between FtsH-His6-Myc and detergent-solubilized SecY was also demonstrated. Although FtsH-bound SecY was processed further for ATP-dependent proteolysis, FtsH-bound PhoA was not. Thus, FtsH association with denatured PhoA is uncoupled from proteolysis. Overproduction of FtsH significantly increased the cytoplasmic localization of the PhoA moiety of a MalF-PhoA hybrid protein, in which a charged residue had been introduced into a transmembrane segment. Thus, denatured PhoA binding of FtsH may also occur in vivo.     

Characterization and Purification of Polydisperse Reconstituted Lipoproteins and Nanolipoprotein Particles

Heterogeneity is a fact that plagues the characterization and application of many self-assembled biological constructs. The importance of obtaining particle homogeneity in biological assemblies is a critical goal, as bulk analysis tools often require identical species for reliable interpretation of the results—indeed, important tools of analysis such as x-ray diffraction typically require over 90% purity for effectiveness. This issue bears particular importance in the case of lipoproteins. Lipid-binding proteins known as apolipoproteins can self assemble with liposomes to form reconstituted high density lipoproteins (rHDLs) or nanolipoprotein particles (NLPs) when used for biotechnology applications such as the solubilization of membrane proteins. Typically, the apolipoprotein and phospholipids reactants are self assembled and even with careful assembly protocols the product often contains heterogeneous particles. In fact, size polydispersity in rHDLs and NLPs published in the literature are frequently observed, which may confound the accurate use of analytical methods. In this article, we demonstrate a procedure for producing a pure, monodisperse NLP subpopulation from a polydisperse self-assembly using size exclusion chromatography (SEC) coupled with high resolution particle imaging by atomic force microscopy (AFM). In addition, NLPs have been shown to self assemble both in the presence and absence of detergents such as cholate, yet the effects of cholate on NLP polydispersity and separation has not been systematically examined. Therefore, we examined the separation properties of NLPs assembled in both the absence and presence of cholate using SEC and native gel electrophoresis. From this analysis, NLPs prepared with and without cholate showed particles with well defined diameters spanning a similar size range. However, cholate was shown to have a dramatic affect on NLP separation by SEC and native gel electrophoresis. Furthermore, under conditions where different sized NLPs were not sufficiently separated or purified by SEC, AFM was used to deconvolute the elution pattern of different sized NLPs. From this analysis we were able to purify an NLP subpopulation to 90% size homogeneity by taking extremely fine elutions from the SEC. With this purity, we generate high quality NLP crystals that were over 100 μm in size with little precipitate, which could not be obtained utilizing the traditional size exclusion techniques. This purification procedure and the methods for validation are broadly applicable to other lipoprotein particles.     

The Optimal Sizing of HSS Database in IMS

Our paper deals with the architecture and standards of IP the multimedia subsystem from next generation networks point of view. It describes the structure of the home subscriber server database and then derives equations for sizing of its load in the proposed scenarios. We have used two methods for the database sizing—Erlang C formula and Markov chains. Finally, the achieved results and used methods are evaluated and discussed.